Caltech researchers have used a state-of-the-art imaging technique to capture detailed 3D views of the complex mobile nanomachinery in bacteria for the first time.

Grant Jensen, a professor of biophysics and biology at Caltech and an investigator with the Howard Hughes Medical Institute (HHMI), and his colleagues used a technique called electron cryotomography to capture 3D images of intact cells with a resolution that ranges from 2 to 5 nanometers (for comparison, a whole cell can be several thousand nanometers in diameter).

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In a paper published in the March 11 issue of the journal Science, the researchers explain they used an advanced technique to analyze bacteria-cell motility machinery — a structure called the type IVa pilus machine (T4PM). This machine enables a bacterium to move through its environment in a manner similar to how Spider-Man travels between skyscrapers.

The T4PM assembles a long fiber (the pilus) that attaches to a surface like a grappling hook and subsequently retracts, thus pulling the cell forward.

First, the researchers froze the bacteria cells instantaneously to prevent water molecules from rearranging to form ice crystals. That locked the cells in place without damaging their structure. Then, using a transmission electron microscope, the researchers imaged the cells from different angles, producing a series of 2-D images. They then digitally reconstructed these images into a 3-D picture of the cell’s structures (like creating a CT scan). Jensen’s laboratory is one of only a few in the entire world that can do this type of imaging.

The researchers found that the bacteria structure is made up of several parts, including a pore on the outer membrane of the cell, four interconnected ring structures, and a stemlike structure. By systematically imaging mutants, each of which lacked one of the 10 T4PM core components, and comparing these mutants with normal M. xanthus cells, they mapped the locations of all 10 T4PM core components, providing insights into pilus assembly, structure, and function.

Strongest motor known in nature — how it works

“The machine lets M. xanthus, a predatory bacterium, move across a field to form a ‘wolf pack’ with other M. xanthus cells, and hunt together for other bacteria on which to prey,” Jensen says. “T4PMs … generate forces as high as 150 pN to pull the cell forward, making T4PMs the strongest molecular motors known,” the authors note.

Another way that bacteria move about their environment is by employing a flagellum — a long whiplike structure that extends outward from the cell. The flagellum is spun by cellular machinery, creating a sort of propeller that motors the bacterium through a substrate (see Tiny swimming ‘biobots’ propelled by heart cells or magnetic fields).

However, cells that must push through the thick mucus of the intestine, for example, need more powerful versions of these motors, compared to cells that only need enough propeller power to travel through a pool of water.

Heavy-duty vs. light-duty bacterial propellers

In a second work, published in an open-access paper in the online early edition of the Proceedings of the National Academy of Sciences (PNAS) on March 14, Jensen and his colleagues used electron cryotomography to study the differences between heavy-duty and light-duty versions of the bacterial propeller. The 3-D images they captured showed that the varying levels of propeller power among several different species of bacteria can be explained by structural differences in these tiny motors.

In order for the flagellum to act as a propeller, structures in the cell’s motor must apply torque — the force needed to cause an object to rotate—to the flagellum. The researchers found that the high-power motors have additional torque-generating protein complexes that are found at a relatively wide radius from the flagellum. This extra distance provides greater leverage to rotate the flagellum, thus generating greater torque. The strength of the cell’s motor was directly correlated with the number of these torque-generating complexes in the cell.

“These two studies establish a technique for solving the complete structures of large macromolecular complexes in situ, or inside intact cells,” Jensen says. “Other structure determination methods, such as X-ray crystallography, require complexes to be purified out of cells, resulting in loss of components and possible contamination. On the other hand, traditional 2-D imaging alone doesn’t let you see where individual protein pieces fit in the complete structure. Our electron cryotomography technique is a good solution because it can be used to look at the whole cell, providing a complete picture of the architecture and location of these structures.”

The work involving the type IVa pilus machinery was published in a Science paper by research scientists at Caltech, the Max Planck Institute for Terrestrial Microbiology, and the University of Utah. The study was funded by the National Institutes of Health (NIH), HHMI, the Max Planck Society, and the Deutsche Forschungsgemeinschaft.

Work involving the flagellum machinery in the second study was published in a PNAS paper. Additional coauthors include collaborators from Imperial College London; the University of Texas Southwestern Medical Center; and the University of Wisconsin–Madison. The study was supported by funding from the UK’s Biotechnology and Biological Sciences Research Council and from HHMI and NIH.

Many bacteria, including important pathogens, move by projecting grappling-hook–like extensions called type IV pili from their cell bodies. After these pili attach to other cells or objects in their environment, the bacteria retract the pili to pull themselves forward. Chang et al. used electron cryotomography of intact cells to image the protein machines that extend and retract the pili, revealing where each protein component resides. Putting the known structures of the individual proteins in place like pieces of a three-dimensional puzzle revealed insights into how the machine works, including evidence that ATP hydrolysis by cytoplasmic motors rotates a membrane-embedded adaptor that slips pilin subunits back and forth from the membrane onto the pilus.

Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, from Campylobacter and Vibrio species. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes.

The nanoparticles start out relatively large (100 nm) (large blue circle, upper left) to enable smooth transport into the tumor through leaky blood vessels. Then, in acidic conditions found close to tumors, the particles discharge “bomblets” (right, small blue circles) just 5 nm in size. Once inside tumor cells, a second chemical step activates the platinum-based drug cisplatin (bottom) to attack the cancer directly. (credit: Emory Health Sciences)

Scientists have devised a triple-stage stealth “cluster bomb” system for delivering the anti-cancer chemotherapy drug cisplatin, using nanoparticles designed to break up when they reach a tumor:

The nanoparticles start out relatively large — 100 nanometers wide — so that they can move through the bloodstream and smoothly transport into the tumor through leaky blood vessels.

As they detect acidic conditions close to tumors, the nanoparticles discharge “bomblets” just 5 nanometers in size to penetrate tumor cells.

Once inside tumor cells, the bomblets release the platinum-based cisplatin, which kills by crosslinking and damaging DNA.

Doctors have used cisplatin to fight several types of cancer for decades, but toxic side effects — to the kidneys, nerves and inner ear — have limited its effectiveness. But in research with three different mouse tumor models*, the researchers have now shown that their nanoparticles can enhance cisplatin drug accumulation in tumor tissues for several types of cancer.

Details of the research — by teams led by professor Jun Wang, PhD, at the University of Science and Technology of China and by professor Shuming Nie, PhD, in the Wallace H. Coulter Department of Biomedical Engineering at Georgia Tech and Emory — were published this week in the journal PNAS.

* When mice bearing human pancreatic tumors were given the same doses of free cisplatin or cisplatin clothed in pH-sensitive nanoparticles, the level of platinum in tumor tissues was seven times higher with the nanoparticles. This suggests the possibility that nanoparticle delivery of a limited dose of cisplatin could restrain the toxic side effects during cancer treatment.

The researchers also showed that the nanoparticles were effective against a cisplatin-resistant lung cancer model and an invasive metastatic breast cancer model in mice. In the lung cancer model, a dose of free cisplatin yielded just 10 percent growth inhibition, while the same dose clothed in nanoparticles yielded 95 percent growth inhibition, the researchers report. In the metastatic breast cancer model, treating mice with cisplatin clothed in nanoparticles prolonged animal survival by weeks; 50 percent of the mice were surviving at 54 days with nanoparticles compared with 37 days for the same dose of free cisplatin.

A principal goal of cancer nanomedicine is to deliver therapeutics effectively to cancer cells within solid tumors. However, there are a series of biological barriers that impede nanomedicine from reaching target cells. Here, we report a stimuli-responsive clustered nanoparticle to systematically overcome these multiple barriers by sequentially responding to the endogenous attributes of the tumor microenvironment. The smart polymeric clustered nanoparticle (iCluster) has an initial size of ∼100 nm, which is favorable for long blood circulation and high propensity of extravasation through tumor vascular fenestrations. Once iCluster accumulates at tumor sites, the intrinsic tumor extracellular acidity would trigger the discharge of platinum prodrug-conjugated poly(amidoamine) dendrimers (diameter ∼5 nm). Such a structural alteration greatly facilitates tumor penetration and cell internalization of the therapeutics. The internalized dendrimer prodrugs are further reduced intracellularly to release cisplatin to kill cancer cells. The superior in vivo antitumor activities of iCluster are validated in varying intractable tumor models including poorly permeable pancreatic cancer, drug-resistant cancer, and metastatic cancer, demonstrating its versatility and broad applicability.